Confluent 3T3-L1 cells have the capacity to acquire many of the morphological and biochemical characteristics of a dipocytes. We reported that during a dipocyte conversion of 3T3- L1 cells, glutamine synthetase (GS) activity and cellular content increase more than 100-fold. In additiona, our data indicate that in 3T3-L1 adipocytes the GS biosynthetic rate is decreased by dibutyryl cAMP or insulin while it is increased by hydrocortisone. The long range goal of the proposed research is to determine the mechanisms by which hormones regulate intermediate metabolism. The specific aims are: 1) to characterize hormonal regulation of GS activity in 3T3 adipocytes, 2) to determine at the level of enzyme synthesis, degradation and covalent modification, the mechanisms by which hormones regulate GS activity, and 3) to determine the effects of hormones and effectors on the activity and cellular content of mRNA coding for GS in 3T3 adipocytes. The mechanisms for observed effects will be evaluated by immunochemical and radiochemical techniques to assess enzyme content, synthesis, degradation and possible covalent modification. GS synthesis rate will be estimated, with pulse-labeled cells, from the ratio between L-[35S] methionine incorporated into immunoprecipitable GS and L-[35S]methionine incorporated into soluble protein. Degradation rate constants will be estimated from the first order disappearance of radioactivity from GS and soluble protein in 3T3 adipocytes previously incubated with L-[35S]methionine. Immunotitration and radioimmunoassay analyses will be performed to assess covalent modification. Functional mRNA coding for GS will be analyzed by cell free translation of polyadenylylated RNA extracted from cultures and isolated by oligo(dT)- cellulose chromatography. GS in translation products will be isolated by immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis. A GS cDNA will be selected from a cDNA "library" prepared with enriched mRNA from hydrocortisone-induced 3T3 adipocytes as template for cDNA synthesis. GS mRNA will be enriched by sucrose density gradient sedimentation, agarose gel electrophoresis, or polysome immunopurification. The cDNA "library", prepared with plasmid pBR322, will be screened for a GS cDNA by differential colony hybridization, by hybridization selected translation, and by hybridization with a synthetic oligonucleotide probe. Cellular content, synthesis, and degradation of GS mRNA will be analyzed by hybridization of cellular mRNA with the specific GS cDNA. GS mRNA will be quantified by dot-blot hybridization, by quantitative hybridization of 5'-end labeled mRNA, and by solution hybridization. Availability of the GS cDNA will allow us to isolate the GS gene and to begin to analyze its structure, organization, and regulatory regions.